Multidimensional studies are ongoing to (a) design and develop recombinant vaccines to tumor-associated gene products; (b) understand host-tumor cell interactions; (c) reveal factors that stimulate immune responses; and (d) design and develop novel immunotherapies for cancer. Patients immune responses to first-generation recombinant vaccines are being analyzed to help design more potent second- and third-generation vaccines and to develop strategies that will enhance T-cell responses. Some ongoing studies include: Design and Development of Recombinant Cancer Vaccines (J. Schlom and K.Y. Tsang, Group Leaders)Gene products that are overexpressed or altered in human carcinomas are being evaluated as targets for recombinant vaccines. These gene products include: (1) carcinoembryonic antigen (CEA), which is overexpressed in gastrointestinal, breast, and lung carcinomas; (2) prostate specific antigen (PSA); and (3) the breast cancer mucin muc-1, which is also overexpressed in lung and other carcinomas. Recombinant vaccinia virus (rV) and recombinant avipox vectors containing the CEA gene are being used in ongoing clinical trials. Vaccination of carcinoma patients has been shown to elicit CEA-specific T-cell responses. Clinical trials that employ rV-PSA as immunogen and that induce PSA-specific T-cell responses are also under way. Yet another clinical trial, one that uses a recombinant vector containing the muc-1 gene, has also been initiated recently. The immunodominant epitopes to which these CEA- and PSA- specific T-cell responses were directed have been identified. A T-cell receptor (TCR) enhancer agonist epitope has also been identified for CEA. Other studies currently in progress involve the design and analysis of second- and third-generation vaccines, including vaccines that contain both a tumor associated antigen (TAA) and one or more T- cell costimulatory molecules. Recent findings have shown that certain cytokines can greatly upregulate patients responses to a tumor antigen when given in conjunction with a recombinant vaccine. T-Cell Costimulation (J. Hodge, Group Leader)Studies on the design and use of recombinant poxviruses to present T-cell costimulatory signals are under way. This approach is potentially useful in both gene therapy and recombinant vaccine development. Recent studies have shown that a recombinant vaccine containing the B7-1, ICAM-1 or other costimulatory molecule gene and a tumor antigen gene leads to enhanced T-cell responses and antitumor activity. Studies are ongoing to design novel vaccines containing multiple costimulatory molecules to better define the principles of optimal costimulation. The activation of a T cell has been shown to require two signals via molecules present on professional antigen presenting cells: signal 1, via a peptide/MHC complex, and signal 2, via a costimulatory molecule. Here, the role of three costimulatory molecules in the activation of T cells was examined. Poxvirus (vaccinia and avipox) vectors were employed because of their ability to efficiently express multiple genes. Murine cells provided with signal 1 and infected with either recombinant vaccinia or avipox vectors containing a TRIad of COstimulatory Molecules (B7-1/ICAM-1/LFA- 3, designated TRICOM) induced the activation of T cells to a far greater extent than cells infected with any one or two costimulatory molecules. Despite this T-cell hyperstimulation using TRICOM vectors, no evidence of apoptosis above that seen using the B7-1 vector was observed. Results employing the TRICOM vectors were most dramatic under conditions of either low levels of first signal or low stimulator cell to T-cell ratios. Experiments employing a four-gene construct also showed that TRICOM recombinants can enhance antigen-specific T-cell responses in vivo. These studies thus demonstrate for the first time the ability of vectors to introduce three costimulatory molecules into cells, thereby activating both CD4+ and CD8+ T-cell populations to levels greater than those achieved with the use of only one or two costimulatory molecules. This new threshold of T-cell activation has broad implications in vaccine design and development. Cytokines as Biological Adjuvants (J. Greiner, Group Leader)Recent research interests have broadened to include the investigation of different cytokines that may augment a T-cell response to a defined tumor antigen. To date, studies have focused on cytokines that enhance T-cell proliferation (i.e., IL-2) and cytokines that act on antigen presentation to T cells (i.e., GM-CSF). For example, IL-2 administration can augment the antitumor efficacy of a recombinant vaccinia virus expressing a TAA. Concomitant GM-CSF administration with an antigen having poor immunogenic properties can enhance the antigen- specific T-cell response. The use of recombinant vectors to express cytokines is also being evaluated. Novel Recombinant Immunoglobulin Forms for Cancer Therapy and Diagnosis (S.V.S. Kashmiri, Group Leader)The overall goal of ongoing research is the design and characterization of recombinant immunoglobulin (Ig) forms for use in both therapeutic and diagnostic applications for a range of human cancers. Emphasis is being placed on the design and translational research of CDR-grafted and CDR-modified forms, domain-deleted Ig forms, and single-chain fusion molecules. Studies are ongoing in experimental models to define metabolic and pharmacokinetic properties, as well as tumor targeting of these recombinant Igs. Studies are also being conducted in which chimeric single-chain antitumor Ig genes and the zeta chain of the TCR are being introduced into T lymphocytes to develop specific, redirected antitumor human T-cell populations.